In the United States, Shiga toxin (Stx)-producing Escherichia coli (STEC) are the most common cause of infectious bloody diarrhea (Rangel et al. (2005) Emerg. Infect. Dis. 11, 603-9) and account for about 110,000 infections per year (Mead et al. (1999) Emerg. Infect. Dis. 5, 607-25). The majority of Stx-mediated disease is attributable to a subset of STEC, the enterohemorrhagic E. coli, which include the prototypic serotype O157:H7. The hemolytic uremic syndrome (HUS) is a serious sequela of STEC (particularly O157:H7) infection that is characterized by hemolytic anemia, thrombic thrombocytopenia and renal failure, especially amongst the most vulnerable patients—children and the elderly. The fatality rate in those who experience HUS is five to ten percent, with the potential for residual kidney and neurological damage among survivors. Therapy for STEC-infections includes supportive care, rehydration and kidney dialysis (Andreoli et al. (2002) Pediatr. Nephrol. 17, 293-8; Klein et al. (2002) J. Pediatr. 141, 172-7; and Tarr et al. (2005) Lancet 365(9464), 1073-86). No interventional therapy or vaccine is currently available. Furthermore, antibiotic treatment is contraindicated due to the increased risk of HUS (Wong et al. (2000) N. Engl. J. Med. 342, 1930-6) that may result from induction of the lytic cycle of the toxin-converting phages that encode Stxs in E. coli. 
There are two main types of Stxs. Members of the first type, Stx produced by S. dysenteriae type 1 and Stx1 produced by E. coli are virtually identical. The second type, Stx2 is also encoded by E. coli; however, it is not cross-neutralized by polyclonal antisera against Stx1, or vice versa (O'Brien et al. (1984) Science 226(4675), 694-6). Variants of each Stx serogroup exist (e.g., Stx1c, Stx1d, Stx2c, Stx2d, Stx2d-activatable, Stx2e, Stx2f) (Melton-Celsa et al. (2005) EcoSal-Escherichia coli and Salmonella: Cellular and Molecular Biology, ASM Press, Chapter 8.7.8) but they remain neutralizable by polyclonal sera to the prototype toxin (Schmitt et al. (1991) Infect Immun 59, 1065-73; Lindgren et al. (1994) Infect. Immun. 62, 623-31). Stxs are complex holotoxins with an AB5 composition. They have an enzymatically active (A) subunit and a binding domain (B) composed of five identical B proteins of about 7.7 kDa each that form a pentamer. The A subunit is a ˜32 kDa protein that is asymmetrically cleaved by trypsin or furin into the A1 subunit (about 27 kDa) and the A2 peptide (about 5 kDa) that remain associated through a disulfide bond. The mature A and B subunits of Stx1 and Stx2 have 55% and 61% identity and 68% and 73% similarity, respectively. Despite the amino acid sequence differences, the crystal structures of the holotoxins are remarkably similar (Fraser et al. (1994) Nat. Struct. Biol. 1, 59-64; Fraser et al. (2004) J. Biol. Chem. 279, 27511-7) and the toxins have the same mode of action. The A1 subunit contains the enzymatically active region, an N-glycosidase that removes an adenosine residue from the 28S rRNA from the 60S ribosome. This alteration halts protein synthesis and kills the intoxicated cell. The A2 peptide traverses the B pentamer to tether the holotoxin together non-covalently. The B pentamer binds the eukaryotic receptor globotriaosyl ceramide (Gb3) or Gb4, as is the case for Stx2e.
Efforts to develop vaccines protective against both Stx types have thus far been frustrating. Stxs are extremely potent and inactivation of the enzymatic activity is necessary to utilize the holotoxins as vaccines. One alternative is to use the B subunits to elicit antibodies that block binding of the B pentamer to the GB3 cellular receptor. This approach has been successful with StxB1 to raise protection against Stx1 challenge, but immununization with the StxB2 subunit is ineffective in protecting against Stx2. Furthermore, passive immunization of mice with anti-StxA2 monoclonal antibody protects mice from the effects of infection with Stx2-producing strains while anti-StxB1 monoclonal antibody is not protective against such a challenge (Wadolkowski et al. (1990) Infect. Immun. 58, 2438-45; Lindgren et al. (1993) Infect. Immun. 61, 383242). However, mice injected with an otherwise lethal dose of Stx1 or Stx2 are protected by passive immunization with anti-StxB1 or anti-StxA2, respectively. The toxicity of the StxA subunits is greatly abrogated without the B pentamer binding domain and there is evidence that vaccines composed of StxA1 and StxA2 offer homologous toxin protection in rabbits (Bielaszewska et al. (1997) Infect. Immun. 65, 2509-16). However, for safety, inactivation of enzymatic activity would be necessary for use of an A subunit vaccine in humans. Subunit vaccines in general are less desirable from the perspective that holotoxin is likely to provide a broader spectrum of protective antibodies than a subunit vaccine.
Protection against toxin-mediated diseases by immunization with toxoid (inactivated holotoxin) vaccines is successful for tetanus and diphtheria. Unfortunately, chemical inactivation of Stxs with formaldehyde or gluteraldehyde is an ill-defined chemical process that can result in residual toxicity (Metz et al. (2003) Vaccine 22, 156-67; Gordon et al. (1992) Infect. Immun. 60, 485-90) or potential distortion of the native holotoxin structure such that neutralizing antibodies are not generated or are of low titer. Some reports in the literature suggest that cross-neutralization has been achieved in animals vaccinated with chemically prepared Shiga toxoids (Bielaszewska et al. (1997) Infect. Immun. 65, 2509-16; Ludwig et al. (2002) Can. J. Microbiol. 48, 99-103); however, the potential for life-threatening toxicity of such a vaccine precludes the use of chemical Stx toxoids in humans. A safer alternative to chemically derived Stx toxoids is the construction of genetic toxoids through the introduction of specific mutations in the Stx A subunit genes to change key amino acids of the enzymatically active domain. Hybrid Stx1 and Stx2 toxins have been made for functional studies of Stxs (Head et al. (1991) J. Biol. Chem. 266, 3617-3621; Weinstein et al. (1989) Infect. Immun. 57, 3743-50; Melton-Celsa et al. (2002) Molecular Microbiology 43, 207-215), including operon fusions allowing A and B subunit expression as a single operon (Weinstein et al., supra). Genetic toxoids of Stx1 or Stx2 that protect animals from subsequent lethal challenges of either Stx1 or Stx2 have previously been made (Gordon et al. (1992) Infect. Immun. 60, 485-90; Ishikawa et al. (2003) Infect. Immun. 71, 3235-9; Wen et al. (2006) Vaccine 24, 1142-8). However, such genetic toxoids are unable to circumvent the lack of cross-neutralization between the Stx1 and Stx2 serogroups and only protect against the Stx group from which they were made. To date, there has been no report in the literature of Stx hybrid toxoids being generated.